Abstract
Human solid tumors and clonal tumor cell lines comprise phenotypically and functionally diverse subsets of cancer cells and also contain stem cell-like cancer cells. Side population (SP) cells, which pump out the fluorescent dye Hoechst 33342 (H33342) via the ABCG2 transporter, define a fraction of adult tissue stem cells in a wide variety of organs. Rare cancer cells with similar H33342 efflux capacity and delimited expression of ABCG2 are present in various types of human tumors, such as breast cancer, lung cancer, and hepatocellular carcinoma. These cancer SP cells display several properties attributable to stem cell-like cancer cells and have been implicated in tumor growth, progression, and metastasis. Here we provide a detailed protocol for the detection of putative cancer stem cells of the SP phenotype in human adherent breast cancer cell cultures.
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References
Wicha MS, Liu S, Dontu G (2006) Cancer stem cells: an old idea—a paradigm shift. Cancer Res 66:1883–1890, discussion 1895–1896
Mackenzie IC (2005) Retention of stem cell patterns in malignant cell lines. Cell Prolif 38:347–355
Wulf GG, Wang RY, Kuehnle I, Weidner D, Marini F, Brenner MK, Andreeff M, Goodell MA (2001) A leukemic stem cell with intrinsic drug efflux capacity in acute myeloid leukemia. Blood 98:1166–1173
Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF (2003) Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci USA 100:3983–3988
Singh SK, Clarke ID, Terasaki M, Bonn VE, Hawkins C, Squire J, Dirks PB (2003) Identification of a cancer stem cell in human brain tumors. Cancer Res 63:5821–5828
Hemmati HD, Nakano I, Lazareff JA, Masterman-Smith M, Geschwind DH, Bronner-Fraser M, Kornblum HI (2003) Cancerous stem cells can arise from pediatric brain tumors. Proc Natl Acad Sci USA 100:15178–15183
Collins AT, Berry PA, Hyde C, Stower MJ, Maitland NJ (2005) Prospective identification of tumorigenic prostate cancer stem cells. Cancer Res 65:10946–10951
O‘Brien CA, Pollett A, Gallinger S, Dick JE (2007) A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature 445:106–110
Dean M, Fojo T, Bates S (2005) Tumour stem cells and drug resistance. Nat Rev Cancer 5:275–284
Balic M, Lin H, Young L, Hawes D, Giuliano A, McNamara G, Datar RH, Cote RJ (2006) Most early disseminated cancer cells detected in bone marrow of breast cancer patients have a putative breast cancer stem cell phenotype. Clin Cancer Res 12:5615–5621
Hill RP (2006) Identifying cancer stem cells in solid tumors: case not proven. Cancer Res 66:1891–1895, discussion 1890
Kondo T, Setoguchi T, Taga T (2004) Persistence of a small subpopulation of cancer stem-like cells in the C6 glioma cell line. Proc Natl Acad Sci USA 101:781–786
Locke M, Heywood M, Fawell S, Mackenzie IC (2005) Retention of intrinsic stem cell hierarchies in carcinoma-derived cell lines. Cancer Res 65:8944–8950
Matsui W, Huff CA, Wang Q, Malehorn MT, Barber J, Tanhehco Y, Smith BD, Civin CI, Jones RJ (2004) Characterization of clonogenic multiple myeloma cells. Blood 103:2332–2336
Phillips TM, McBride WH, Pajonk F (2006) The response of CD24(-/low)/CD44+ breast cancer-initiating cells to radiation. J Natl Cancer Inst 98:1777–1785
Ponti D, Costa A, Zaffaroni N, Pratesi G, Petrangolini G, Coradini D, Pilotti S, Pierotti MA, Daidone MG (2005) Isolation and in vitro propagation of tumorigenic breast cancer cells with stem/progenitor cell properties. Cancer Res 65:5506–5511
Fang D, Nguyen TK, Leishear K, Finko R, Kulp AN, Hotz S, Van Belle PA, Xu X, Elder DE, Herlyn M (2005) A tumorigenic subpopulation with stem cell properties in melanomas. Cancer Res 65:9328–9337
Zhou J, Wulfkuhle J, Zhang H, Gu P, Yang Y, Deng J, Margolick JB, Liotta LA, Petricoin E 3rd, Zhang Y (2007) Activation of the PTEN/mTOR/STAT3 pathway in breast cancer stem-like cells is required for viability and maintenance. Proc Natl Acad Sci USA 104:16158–16163
Resnicoff M, Medrano EE, Podhajcer OL, Bravo AI, Bover L, Mordoh J (1987) Subpopulations of MCF7 cells separated by Percoll gradient centrifugation: a model to analyze the heterogeneity of human breast cancer. Proc Natl Acad Sci USA 84:7295–7299
Ma S, Chan KW, Hu L, Lee TK, Wo JY, Ng IO, Zheng BJ, Guan XY (2007) Identification and characterization of tumorigenic liver cancer stem/progenitor cells. Gastroenterology 132:2542–2556
Ma S, Lee TK, Zheng BJ, Chan KW, Guan XY (2008) CD133+ HCC cancer stem cells confer chemoresistance by preferential expression of the Akt/PKB survival pathway. Oncogene 27:1749–1758
Yin S, Li J, Hu C, Chen X, Yao M, Yan M, Jiang G, Ge C, Xie H, Wan D, Yang S, Zheng S, Gu J (2007) CD133 positive hepatocellular carcinoma cells possess high capacity for tumorigenicity. Int J Cancer 120:1444–1450
Charafe-Jauffret E, Ginestier C, Iovino F, Wicinski J, Cervera N, Finetti P, Hur MH, Diebel ME, Monville F, Dutcher J, Brown M, Viens P, Xerri L, Bertucci F, Stassi G, Dontu G, Birnbaum D, Wicha MS (2009) Breast cancer cell lines contain functional cancer stem cells with metastatic capacity and a distinct molecular signature. Cancer Res 69:1302–1313
Ginestier C, Hur MH, Charafe-Jauffret E, Monville F, Dutcher J, Brown M, Jacquemier J, Viens P, Kleer CG, Liu S, Schott A, Hayes D, Birnbaum D, Wicha MS, Dontu G (2007) ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell 1:555–567
Hirschmann-Jax C, Foster AE, Wulf GG, Nuchtern JG, Jax TW, Gobel U, Goodell MA, Brenner MK (2004) A distinct “side population” of cells with high drug efflux capacity in human tumor cells. Proc Natl Acad Sci USA 101:14228–14233
Patrawala L, Calhoun T, Schneider-Broussard R, Zhou J, Claypool K, Tang DG (2005) Side population is enriched in tumorigenic, stem-like cancer cells, whereas ABCG2+ and ABCG2- cancer cells are similarly tumorigenic. Cancer Res 65:6207–6219
Haraguchi N, Utsunomiya T, Inoue H, Tanaka F, Mimori K, Barnard GF, Mori M (2006) Characterization of a side population of cancer cells from human gastrointestinal system. Stem Cells 24:506–513
Szotek PP, Pieretti-Vanmarcke R, Masiakos PT, Dinulescu DM, Connolly D, Foster R, Dombkowski D, Preffer F, Maclaughlin DT, Donahoe PK (2006) Ovarian cancer side population defines cells with stem cell-like characteristics and Mullerian Inhibiting Substance responsiveness. Proc Natl Acad Sci USA 103:11154–11159
Chiba T, Kita K, Zheng YW, Yokosuka O, Saisho H, Iwama A, Nakauchi H, Taniguchi H (2006) Side population purified from hepatocellular carcinoma cells harbors cancer stem cell-like properties. Hepatology 44:240–251
Christgen M, Ballmaier M, Bruchhardt H, von Wasielewski R, Kreipe H, Lehmann U (2007) Identification of a distinct side population of cancer cells in the Cal-51 human breast carcinoma cell line. Mol Cell Biochem 306:201–212
Zhou S, Schuetz JD, Bunting KD, Colapietro AM, Sampath J, Morris JJ, Lagutina I, Grosveld GC, Osawa M, Nakauchi H, Sorrentino BP (2001) The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype. Nat Med 7:1028–1034
Scharenberg CW, Harkey MA, Torok-Storb B (2002) The ABCG2 transporter is an efficient Hoechst 33342 efflux pump and is preferentially expressed by immature human hematopoietic progenitors. Blood 99:507–512
Diestra JE, Scheffer GL, Catala I, Maliepaard M, Schellens JH, Scheper RJ, Germa-Lluch JR, Izquierdo MA (2002) Frequent expression of the multi-drug resistance-associated protein BCRP/MXR/ABCP/ABCG2 in human tumours detected by the BXP-21 monoclonal antibody in paraffin-embedded material. J Pathol 198:213–219
Camargo FD, Ramos CA, Goodell MA (2004) Handbook of stem cells. Academic, Orlando, FL, pp 329–336
Goodell MA, Brose K, Paradis G, Conner AS, Mulligan RC (1996) Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med 183:1797–1806
Goodell MA, Rosenzweig M, Kim H, Marks DF, DeMaria M, Paradis G, Grupp SA, Sieff CA, Mulligan RC, Johnson RP (1997) Dye efflux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species. Nat Med 3:1337–1345
Challen GA, Little MH (2006) A side order of stem cells: the SP phenotype. Stem Cells 24:3–12
Dontu G, Abdallah WM, Foley JM, Jackson KW, Clarke MF, Kawamura MJ, Wicha MS (2003) In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes Dev 17:1253–1270
Behbod F, Xian W, Shaw CA, Hilsenbeck SG, Tsimelzon A, Rosen JM (2006) Transcriptional profiling of mammary gland side population cells. Stem Cells 24:1065–1074
Alvi AJ, Clayton H, Joshi C, Enver T, Ashworth A, Vivanco MM, Dale TC, Smalley MJ (2003) Functional and molecular characterisation of mammary side population cells. Breast Cancer Res 5:R1–R8
Kim M, Turnquist H, Jackson J, Sgagias M, Yan Y, Gong M, Dean M, Sharp JG, Cowan K (2002) The multidrug resistance transporter ABCG2 (breast cancer resistance protein 1) effluxes Hoechst 33342 and is overexpressed in hematopoietic stem cells. Clin Cancer Res 8:22–28
Setoguchi T, Taga T, Kondo T (2004) Cancer stem cells persist in many cancer cell lines. Cell Cycle 3:414–415
Zheng X, Shen G, Yang X, Liu W (2007) Most C6 cells are cancer stem cells: evidence from clonal and population analyses. Cancer Res 67:3691–3697
Burkert J, Otto WR, Wright NA (2008) Side populations of gastrointestinal cancers are not enriched in stem cells. J Pathol 214:564–573
Adamski D, Mayol JF, Platet N, Berger F, Herodin F, Wion D (2007) Effects of Hoechst 33342 on C2C12 and PC12 cell differentiation. FEBS Lett 581:3076–3080
Bar EE, Chaudhry A, Lin A, Fan X, Schreck K, Matsui W, Vescovi AL, Piccirillo S, Dimeco F, Olivi A, Eberhart CG (2007) Cyclopamine-mediated hedgehog pathway inhibition depletes stem-like cancer cells in glioblastoma. Stem Cells 25:2524–2533
Zen Y, Fujii T, Yoshikawa S, Takamura H, Tani T, Ohta T, Nakanuma Y (2007) Histological and culture studies with respect to ABCG2 expression support the existence of a cancer cell hierarchy in human hepatocellular carcinoma. Am J Pathol 170:1750–1762
Ho MM, Ng AV, Lam S, Hung JY (2007) Side population in human lung cancer cell lines and tumors is enriched with stem-like cancer cells. Cancer Res 67:4827–4833
Wang J, Guo LP, Chen LZ, Zeng YX, Lu SH (2007) Identification of cancer stem cell-like side population cells in human nasopharyngeal carcinoma cell line. Cancer Res 67:3716–3724
Mitsutake N, Iwao A, Nagai K, Namba H, Ohtsuru A, Saenko V, Yamashita S (2007) Characterization of side population in thyroid cancer cell lines: cancer stem-like cells are enriched partly but not exclusively. Endocrinology 148:1797–1803
Woodward WA, Chen MS, Behbod F, Alfaro MP, Buchholz TA, Rosen JM (2007) WNT/beta-catenin mediates radiation resistance of mouse mammary progenitor cells. Proc Natl Acad Sci USA 104:618–623
Harris MA, Yang H, Low BE, Mukherje J, Guha A, Bronson RT, Shultz LD, Israel MA, Yun K (2008) Cancer stem cells are enriched in the side population cells in a mouse model of glioma. Cancer Res 68:10051–10059
Engelmann K, Shen H, Finn OJ (2008) MCF7 side population cells with characteristics of cancer stem/progenitor cells express the tumor antigen MUC1. Cancer Res 68:2419–2426
Yin L, Castagnino P, Assoian RK (2008) ABCG2 expression and side population abundance regulated by a transforming growth factor beta-directed epithelial-mesenchymal transition. Cancer Res 68:800–807
Kabashima A, Higuchi H, Takaishi H, Matsuzaki Y, Suzuki S, Izumiya M, Iizuka H, Sakai G, Hozawa S, Azuma T, Hibi T (2009) Side population of pancreatic cancer cells predominates in TGF-beta-mediated epithelial to mesenchymal transition and invasion. Int J Cancer 124:2771–2779
Xu JX, Morii E, Liu Y, Nakamichi N, Ikeda J, Kimura H, Aozasa K (2007) High tolerance to apoptotic stimuli induced by serum depletion and ceramide in side-population cells: high expression of CD55 as a novel character for side-population. Exp Cell Res 313:1877–1885
Clarke RB, Spence K, Anderson E, Howell A, Okano H, Potten CS (2005) A putative human breast stem cell population is enriched for steroid receptor-positive cells. Dev Biol 277:443–456
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Christgen, M., Ballmaier, M., Lehmann, U., Kreipe, H. (2012). Detection of Putative Cancer Stem Cells of the Side Population Phenotype in Human Tumor Cell Cultures. In: Dwek, M., Brooks, S., Schumacher, U. (eds) Metastasis Research Protocols. Methods in Molecular Biology, vol 878. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-854-2_13
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DOI: https://doi.org/10.1007/978-1-61779-854-2_13
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